In order to facilitate discussion of known fluorescent entities, their application and the manner in which the fluorescent entities in this invention distinguish from known systems, reference will be made to several scientific articles throughout the specification. To facilitate such reference the articles have been listed by number as follows:
1. Diamandis, E. P. Clin. Biochem. 1988, 21, 139-150. PA1 2. Diamandis, E. P. Clin. Chim. Acta. 1990, 194, 19-50. PA1 3. Diamandis, E. P.; Christopoulos, T. K. Anal. Chem. 1990, 62, 11 49A-11 57A. PA1 4. Soini, E.; Lovgren, T. CRC Crit. Rev. Anal. Chem. 1987, 18, 105-154. PA1 5. Hemmila, I.; Dakubu, S.; Mukkala V. M.; Siitari, H.; Lovgren, T. Anal. Biochem. 137, 335-343. PA1 6. Soini, E.; Kojola, H. Clin. Chem. 1983, 29, 15-68. PA1 7. Evangelista, R. A.; Pollak, A.; Allore, B.; Templeton, E. F.; Morton, R. C.; Diamandis, E. P. Clin. Biochem. 1988, 21, 173-178. PA1 8. Diamandis, E. P.; Morton, R. C.; J. Immunol. Method 1988, 12, 43-52. PA1 9. Bailey, M. P.; Rocks, B. F.; Riley, C. Analyst 1984, 109, 1449-1450. PA1 10. Oser, A.; Gollasius, M.; Valet, G. Anal. Biochem. 1990, 191, 295-301. PA1 11. Hemmila, I.; I Hoittinen, S.; Pettersson, K.; Lovgren, T. Clin. Chem. 1987, 33, 2281-2283. PA1 12. Sinha, A. P. B. Spectrosc. Inorg. Chem. 1971, 2, 255-265. PA1 13. Kallistratos, G. Chimica Chronica New Series, 1982, 11, 249-266. PA1 14. Diamandis, E. P.; Morton, R. C.; Reichstein, E.; Khosravi, M. J. Anal. Chem. 1989, 61, 48-53. PA1 15. Morton, R. C.; Diamandis, E. P. Anal. Chem. 1990, 62, 1841-1845. PA1 16. Diamandis, E. P. Clin. Chem. 1991, 37, 1486-1491. PA1 17. Evangelists, R. A.; Pollak, A.; Gudgin-Templeton, E. F. Anal. Biochem. 1991, 197, 213-224. PA1 18. Christopoulos, T. K.; Diamandis, E. P. Anal. Chem. 1992, (in press). PA1 19. Papanastasiou-Diamandi, A.; Christopoulos, T. K.; Diamandis, E. P. Clin. Chem. 1992 (in press). PA1 20. Papanastasiou-Diamandi, A.; Bhayana, V.; Diamandis, E. P. Ann. Clin. Biochem. 1989, 26, 238-243.
Fluorescent lanthanide metal chelates and in particular those for europium and terbium chelates have significant commercial application because of their use as labels in highly sensitive time-resolved fluorometric immunoassays (1-4). The first commercially available time-resolved fluorescence immunoassay system, Delfia.TM. (available by Pharmacia-LKB, Sweden) uses Eu.sup.3+ as immunological label (5,6). A second-generation time-resolved fluorometric immunoassay system, FlAgen.TM. (available by CyberFluor Inc., Toronto, Canada, the applicant in this application) uses the europium chelator 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) as immunological label (7.8). These two systems, along with the principles of time-resolved fluorometry and its application to immunoassay and other bioanalytical techniques have been investigated in detail (1-4).
Recently, Tb.sup.3+ and its chelates have been used as immunological and nucleic acid labels (9,10). In general, Tb.sup.3+ is inferior to Eu.sup.3+ in terms of detectability. However, Eu.sup.3+ and Tb.sup.3+ can be used simultaneously for dual analyte assays (11).
The mechanism of fluorescence of the Eu.sup.3+ and Tb.sup.3+ chelates has been described (1-4). These two ions, when excited by radiation, emit very weak metal-ion fluorescence which is not analytically useful. The fluorescence is dramatically enhanced when the metal-ion forms a chelate with appropriate organic ligands. An important property of these chelates is that the radiation is absorbed at a wavelength characteristic of the ligand and is emitted as a line spectrum characteristic of the metal-ion. This is due to an intramolecular energy transfer from the ligand to the central metal-ion (12). In general, it is difficult to predict theoretically which organic molecules-ligands can form highly fluorescent complexes with Eu.sup.3+ and Tb.sup.3+. Some classes of compounds i.e. the diketones, tetracyclines, phenanthrolines, acetylene derivatives, five-membered heterocyclic ring derivatives, benzoic acid derivatives, biphenyl derivatives, pyridine derivatives, pyrimidine and pyrazine derivatives, di- and tripyridyl derivatives, quinoline derivatives, aza-uracil and purine derivatives and phosphorimido-derivatives have been identified as fluorogenic ligands of Eu.sup.3+ and/or Tb.sup.3+ (13).
In time-resolved fluorometric immunoassays, it is desirable to use a chelate-label which can be detected down to the subpicomolar range (5). Alternatively, multiple labelling strategies can be used in order to achieve subplcomolar analyte sensitivity (1 and 4-16). More recently, there have been efforts to combine time-resolved fluorometric immunoassay with enzymatic catalysis. In one version of this approach, the primary immunological label is alkaline phosphatase (ALP); its acid (FSAP). The nonhydrolysed ester (FSAP) and the hydrolysed ester (FSA) have different behaviour in Tb.sup.3+ -EDTA solutions. When FSA is added to an aqueous alkaline solution containing Tb.sup.3+ -EDTA, a mixed complex is formed which emits long-lived fluorescence characteristic of Tb.sup.3+. FSA is an appropriate ligand for energy transfer to the metal-ion. An intact hydroxyl group on the FSA molecule is essential for these highly fluorescent mixed complexes to be formed. FSAP does not form any fluorescent complexes with Tb.sup.3+ -EDTA. In a heterogeneous immunoassay design, ALP activity can be monitored with use of FSAP as substrate, by measuring the released FSA after adding an alkaline solution of Tb.sup.3+ -EDTA (17). This system has been used for the highly sensitive and rapid quantification of alpha-fetoprotein and thyrotropin in serum (18,19).